To determine risk for drug-resistant malaria parasites entering
Madagascar from Comoros Islands, we screened travelers. For the 141
Plasmodium falciparum isolates detected by real-time PCR, frequency of
mutant alleles of genes associated with resistance to chloroquine and
pyrirnethamine was high. International-level antimalarial policy and a
regional antimalarial forum are needed.

**********

In the southwestern Indian Ocean, the epidemiologic features of
malaria and antimalarial drug resistance differ considerably between
islands that are very close geographically. Malaria remains a major
public health problem in Madagascar and the Comoros Islands, whereas the
situation is different on other nearby islands. Chloroquine resistance
ranges from moderate in Madagascar (1-3) to high in the Comoros Islands
(4,5), whereas pyrimethamine resistance is absent in Madagascar (3,6)
but present at high levels in the Comoros Islands (5). The paradoxical
situation of resistance to antimalarial drugs and prevalence of
mutant-type parasites (for the Plasmodium falciparum chloroquine
resistance transporter [pfcrt] gene, 90% of 76T alleles in the Comoros
Islands vs. 3% in Madagascar; for the P. falciparum dihydrofolate
reductase [dhfr] gene, 69% of 108N alleles in the Comoros Islands vs. 0%
in Madagascar) (2,5,7) in such close geographic proximity led us to
perform this study.

Historically, the Comoros Islands and Madagascar have been linked
by human travel, and the importation of pathogens has already been
documented, in the cholera epidemic of 1998-1999 (8) and in more recent
outbreaks of arbovirus infection (9,10). Most travel between the Comoros
Islands and Madagascar occurs through the seaport and airport of
Mahajanga, the main city on the northwestern coast of Madagascar. To
improve the monitoring of antimalarial drug resistance in Madagascar, we
assessed the frequency of P. falciparum mutant alleles of genes
associated with resistance to chloroquine (pfcrt and P. falciparum
multidrug resistance 1 [pfmdr-1] gene) and pyrimethamine (dhfr) among
travelers entering Madagascar from the Comoros Islands.

The Study

The study was performed from March to July 2006, in the seaport and
the airport of Mahajanga, on the northwest coast of Madagascar (Figure
1). These sites are the main communications crossroads between the
Comoros Islands and Madagascar. The study was approved by the National
Ethics Committee of the Ministry of Health and Family Planning of
Madagascar.

All travelers from the Comoros Islands who consented to participate
on arrival in Madagascar, regardless of their age, sex, nationality, and
presence or absence of symptoms, were enrolled in the study. For each
participant,, a questionnaire was filled out and a finger-prick blood
sample was collected. Rapid diagnostic tests (OptiMAL-IT, DiaMed AG,
Cressier sur Morat, Switzerland) and thick/thin blood smears were
performed in the field. Patients with a positive rapid test result were
promptly treated with an artesunate and amodiaquine combination
(Arsucam, Sanofi-Aventis, Paris, France), according to Madagascar's
national malaria policy.

Thick/thin blood smears were stained and analyzed by an experienced
technician, without reference to rapid test results. A minimum of 200
consecutive fields were counted for each thick blood film before a slide
was classified as negative. The number of parasites in thick blood films
was determined per 200 or 500 leukocytes, assuming 8,000
leukocytes/[micro]L of blood. Thin blood smears were also examined for
other Plasmodium spp.

Parasite DNA was extracted from blood samples by using the
phenol/chloroform method. P. falciparum carriers were detected by
real-time PCR in a RotorGene 3000 thermocycler (Corbett Life Science,
Sydney, New South Wales, Australia), as described by Mangold et al.
(11). PCR and restriction fragment length polymorphism analyses were
performed for 3 genes (codon 108 of dhfr, codon 76 of pfcrt, and codon
86 of pfmd-1) for the detection of mutant alleles. (Detailed
descriptions of these methods are available from
http://medschool.umaryland.edu/cvd/ plowe.html.) Laboratory strains of
P. falciparum were used as controls (positive and negative) and included
in all PCR and enzyme digestion procedures (DNA from the W2, HB3, and
3D7 reference strains from the Malaria Research and Reference Reagent
Resource Center, Division of Microbiology and Infectious Diseases,
National Institute of Allergy and Infectious Disease, National
Institutes of Health, Manassas, VA, USA). Statistical analyses were
performed by using SPSS software (SPSS Inc., Chicago, USA). Odds ratios
were calculated from logistic regression parameter estimates, and p
values were determined with the significance level set at p [less than
or equal to] 0.05.

[FIGURE 1 OMITTED]

Among the 1,130 travelers registered on arrival in Mahajanga, 947
agreed to participate in the study (crude participation rate 83.8%). The
baseline characteristics of the enrolled travelers are given as a
function of nationality (Comorian or Malagasy) in the Table. The
frequency of P. falciparum carriers was 0.5% (5/947) according to rapid
diagnostic test, 3.2% (30/947) according to microscopy, and 14.9%
(141/947) according to real-time PCR.

For the 141 P. falciparum isolates detected by real-time PCR, the
frequency of the mutant alleles of genes associated with resistance to
chloroquine (pfcrt andpfmdr-1) and pyrimethamine (dhfr) was 80.1%
(113/141) for the 76T mutant allele ofthepfcrt gene, 99.3% (140/141 )
for the 86Y mutant allele of the pfmdr-1 gene, and 95.0% (134/141) for
the 108N allele of the dhfr gene. More detail is provided in the Table.
Univariate analysis of risk factors associated with the carriage of P.
falciparum mutant alleles showed that for Comorian travelers, only a
history of travel in Africa in the past 3 months was identified as
significant (odds ratio 2.29, 95% confidence interval 1.27-4.13,
p<0.01). We used these data to generate a map assessing the potential
risk for the spread of P. falciparum mutant-type alleles in Madagascar
(Figure 2).

Conclusions

Despite some methodologic limitations (limited study period,
limited number of passengers screened, taking into account only
registered travelers), this study provides the first, to our knowledge,
direct measurement of parasite movement between the Comoros Islands and
Madagascar. This study thus enables an assessment of the potential
threat of P. falciparum mutant allele parasites being introduced into
Madagascar.

First, we noted that for detection of P. falciparum carriers,
real-time PCR was 4.6 times more sensitive than microscopy and 30 times
more sensitive than rapid diagnostic testing based on parasite lactate
dehydrogenase detection, according to the threshold detection level of
the techniques used (11,12). The results suggest that most of the P.
falciparum carriers had low-level parasitemia. Second, most of the
imported parasites carried resistance-associated mutations, consistent
with the frequency of mutant forms of P. falciparum circulating in the
Comoros Islands (4,5). Third, according to the places in which the
Comorian travelers stayed and the places in which the Malagasy travelers
lived, the potential area of antimalarial drug--resistant parasite
spread was located in the northwestern area, which has a transmission
season of >6 months per year (13).

[FIGURE 2 OMITTED]

On the basis of our findings, we suggest that antimalarial drug
policy should be formulated at an international, rather than a national,
level. Given that the Malagasy government's goal is moving toward
malaria elimination, international considerations in antimalarial policy
would avoid the possibility of a coherent national policy being annulled
by mutations originating in or spreading through neighboring countries.
For example, the introduction of high-level chloroquine or pyrimethamine
resistance from the Comoros Islands could compromise the use of the
artesunate and amodiaquine combination as a first-line treatment for
uncomplicated falciparum malaria or the use of sulfadoxine-pyrimethamine
for intermittent preventive treatment in pregnant women and render these
strategies useless.

In conclusion, we suggest the creation, as soon as possible, of a
regional antimalarial forum in the Indian Ocean, similar to the East
African Network for Monitoring Antimalarial Treatment (www.eanmat.org).
Such a forum would enable the countries of the region to share national
information on antimalarial drug efficacy, such as the prevalence of
drug resistance molecular markers, and to debate proposed changes in
national policy.

Acknowledgments

We thank the travelers for their participation in this study and
the trainee doctors for their valuable assistance.

This study received financial support from the French Government
through the FSP/RAI 2001-168 project (French Ministry of Foreign
Affairs) and from the Global Fund to Fight AIDS, Tuberculosis and
Malaria, round 3 (Community Action to Roll Back Malaria, grant no.
MDG-304-G05-M).

Dr Menard is head of malaria unit research in the Institut Pasteur
of Madagascar. His current research interest is surveillance of
antimalarial drug resistance in Madagascar.